Engineering genetically controlled microbial consortia

Author

Chen, Ye

Date

2016-06-24

Advisor

Bennett, Matthew; Beckingham, Kathleen

Degree

Doctor of Philosophy

Abstract

To date, the majority of synthetic gene circuits have been constructed to operate within single, isogenic cellular populations. Two of the toughest challenges for synthetic biologists to achieve complicated multi-strain systems are the limited choice of inducible signals and tuning regulatory components within a gene circuit to elicit desired outputs. Here, we describe a method that allows one to tune the dynamic range in a motif based construction of promoters with regulatory elements. To do this, we first assembled and then tested a library of promoters using different -10 and -35 sites taken from endogenous promoters within Escherichia coli. By mixing and matching the -10 and -35 sites, we were able to create a large number of ligand-inducible promoters exhibiting a wide variety of dynamic ranges. Using this method, we developed an orthogonal, tightly controlled two-signaling system. Then, we used two genetically distinct populations of Escherichia coli and this signaling system to engineer a bacterial consortium that exhibits robust oscillations in gene transcription. When co-cultured in a microfluidic device, the two strains form coupled positive and negative feedback loops at the population-level. The interacting strains exhibit robust, synchronized oscillations that are absent if either strain is cultured in isolation. We further used a combination of mathematical modeling and targeted genetic perturbations to better understand the roles of circuit topology and regulatory promoter strengths in generating and maintaining these oscillations. We found that the dual-feedback topology was robust to changes in promoter strengths and fluctuations in the population ratio of the two strains. These findings demonstrate that one can program population-level dynamics through the genetic engineering of multiple cooperative strains and point the way towards engineering complex synthetic tissues and organs with multiple cell types.